A major problem in developmental neurobiology is the question of how nerve cells are able to make specific connections. This capability depends on the ability of axonal growth cones to navigate accurately over prescribed routes. The purpose of this project is to understand, in a particularly advantageous "model" system, how such navigation occurs. In limb buds of embryonic grasshoppers, the first "pioneer" neurons project axons on a stereotyped path. We are studying what the guidance features are which mark the route, how these features are located by growth cones, and how location of the guidance features results in steering of the growth cones in appropriate directions. Currently available evidence indicates that the guidance features are a chain of "guidepost" cells whose location marks the route, and suggests that these cells may be located by undirected exploration of the cellular landscape by filopodia extended from growth cones. In the next project period, we intend to solidify the evidence for the guidepost hypothesis, to test the filopodial exploration hypothesis, and to explore certain hypotheses for growth cone steering. The normal behavior of these in vivo growth cones can be observed with fluorescently-tagged antibodies. We plan to extend to observations from fixed tissue with monitoring of growing cells and with pulse-labeling. The system can be experimentally manipulated by deletion of specific pioneer neurons or guidepost cells with photoinactivation of dye-injected cells, or with a UV microbeam. We hope to extend manipulation with laser-deletion of filopodia, and with cell-culture techniques which will enable tests of cell-surface properties, the role of adhesion, and the alternative hypotheses of filopodial exploration or diffusible factors for locating guidepost cells. Understanding how nerve cells make specific connections is medically important both for analysis of neuro-pathological conditions, and for restoring normal function after neural trauma.